Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
1. The Field of the Invention
This invention relates to the medical arts. In particular, it relates to a method of detecting a genetic predisposition to systemic lupus erythematosus.
2. Discussion of the Related Art
Systemic lupus erythematosus (SLE), commonly known as lupus, is an autoimmune rheumatic disease characterized by deposition in tissues of autoantibodies and immune complexes leading to tissue injury (B. L. Kotzin, Systemic lupus erythematosus, Cell 85:303–06 [1996]). In contrast to autoimmune diseases such as multiple sclerosis and type 1 diabetes mellitus, SLE potentially involves multiple organ systems directly, and its clinical manifestations are diverse and variable. (Reviewed by B. L. Kotzin and J. R. O'Dell, Systemic lupus erythematosus, In: Samler's Immunologic Diseases, 5th ed., M. M. Frank et al., eds., Little Brown & Co., Boston, pp. 667–97 [1995]).
For example, some patients may demonstrate primarily skin rash and joint pain, show spontaneous remissions, and require little medication. At the other end of the spectrum are patients who demonstrate severe and progressive kidney involvement that requires therapy with high doses of steroids and cytotoxic drugs such as cyclophosphamide. (B. L. Kotzin [1996]). The serological hallmark of SLE, and the primary diagnostic test available until now, is elevated serum levels of IgG antibodies to constituents of the cell nucleus, such as double-stranded DNA (dsDNA), single-stranded DNA (ss-DNA), and chromatin. Among these autoantibodies, IgG anti-dsDNA antibodies play a major role in the development of lupus glomerulonephritis (GN). (B. H. Hahn and B. Tsao, Antibodies to DNA, In: Dubois Lupus Erythematosus, 4th ed., D. J. Wallace and B. Hahn, eds., Lea and Febiger, Philadelphia, pp. 195–201 [1993]; Ohnishi et al., Comparison of pathogenic and nonpathogenic murine antibodies to DNA: Antigen binding and structural characteristics, Int. Immunol. 6:817–30 [1994]). Glomerulonephritis is a serious condition in which the capillary walls of the kidney's blood purifying glomeruli become thickened by accretions on the epithelial side of glomerular basement membranes. The disease is often chronic and progressive and may lead to eventual renal failure.
Mechanisms by which autoantibodies are induced remain unclear. Chromatin and/or nucleosomes, released by apoptotic cells in SLE, may become autoantigens that induce autoimmune responses, including antibodies to dsDNA. (R. W. Burlingame et al., The central role of chromatin in autoimmune responses to histones and DNA in systemic lupus erythematosus, J. Clin. Invest. 94:184–92 [1994]; C. Mohan et al., Nucleosome: a major immunogen for pathogenic autoantibody-inducing T cells of lupus, J. Exp. Med. 177:1367–81 [1993]; D. A. Bell et al., Immunogenic DNA-related factors. Nucleosomes spontaneously released from normal murine lymphoid cells stimulate proliferation and immunoglobulin synthesis of normal mouse lymphocytes, J. Clin. Invest. 85:1487–96 [1990]).
Cumulative studies suggest that interaction of multiple genes and environmental factors result in susceptibility to SLE, as is true for many multifactorial complex human diseases. (F. C. Arnett, Jr., The genetics of human lupus, In: Dubois' Lupus Erythematosus, 5th ed., D. J. Wallace and B. Hahn, eds., Williams and Wilkins, Baltimore, pp. 77–117 [1997]; T. J. Vyse and B. L. Kotzin, Genetic susceptibility to systemic lupus erythematosus, Ann. Rev. Immunol. 16:261–92 [1998]). Although SLE can occur at nearly any age, it primarily affects women of childbearing age; the female to male ratio is greatest (>8:1) for patients presenting between ages 15 to 50 years; incidence rates for patients and studies in certain animal models support a role for estrogens enhancing disease development, and androgens protecting against it. (B. L. Kotzin [1996]). Although rare among males, SLE may be linked in males with functional hypoandrogenism and a higher than normal estradiol/testosterone ratio. (J. F. Sequeira et al., Systemic lupus erythematosus: sex hormones in male patients, Lupus 2(5):315–17 [1993]).
It appears that underlying genetic factors exert the greatest influence on autoantibody production and on predisposition to SLE, as studies of populations, segregation of disease in families, and twin concordance rates have consistently demonstrated. The prevalence of SLE in the general population is approximately 15–50 in 100,000. (M. D. Hochberg, The epidemiology of systemic lupus erythematosus, In: Dubois' Lupus Erythematosus, 5th ed., D. J. Wallace and B. Hahn, eds., Williams and Wilkins, Baltimore, pp. 49–65 [1997]). The relatively high incidence (10–16%) of more than one case in a family has suggested a genetic basis for SLE. The concordance rate of SLE in monozygotic twins (24%–57%) is approximately ten times higher than the rate in dizygotic twins (2–5%). (F. C. Arnett, Jr. [1997]; M. D. Hochberg [1997]). Based on these epidemiological studies, the relative risk for siblings of SLE patients compared to the general population, λs, is at least 40-fold. (See, N. Risch, Assessing the role of HLA-linked and unlinked determinants of disease, Am. J. Hum. Genet. 40:1–14 [1987]).
The genetic basis for SLE in humans is complex, with an unknown but non-Mendelian mode of inheritance. This complexity has impeded the development of a reliable and predictive genetic test for SLE until the present invention.
Many investigators have reported that certain human MHC class II alleles (HLA-DR and/or DQ but not DP) and certain class III genes (C2, C4, TNFα, and HSP70-2 alleles) confer susceptibility to SLE in most ethnic groups studied. (F. C. Arnett, Jr., The genetics of human lupus, In: Dubois' Lupus Erythematosus, 5th ed., D. J. Wallace and B. Hahn, eds., Williams and Wilkins, Baltimore, pp. 77–117[1997]; T. J. Vyse and B. L. Kotzin, Genetic susceptibility to systemic lupus erythematosus, Ann. Rev. Immunol. 16:261–92 [1998]). Among the other non-MHC genes that have been associated with SLE, evidence for homozygous deficiency of C1q predisposing to SLE is particularly compelling, including the observation that 90% of such individuals have SLE and C1q knockout mice display an SLE-like phenotype. (M. Botto et al., Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies, Nat. Genet. 19:56–59 [1998]; P. Bowness et al., Hereditary C1q deficiency and systemic lupus erythematosus, Quart. J. Med. 87:455–64 [1994]).
In addition, polymorphisms in many genes encoding molecules with relevant immunological functions have been studied most frequently by the case-control approach, including T-cell receptor α and β chains, immunoglobulin allotypes, FcγRIIa, FcgRIIIa, IL-6, IL-10, Bcl-2, mannose-binding protein (or lectin), as well as deletion of specific variable gene segments of immunoglobulin genes. (F. C. Arnett, Jr. [1997]; T. J. Vyse and B. L. Kotzin [1998]; J. Wu et al., A novel polymorphism of FcγRIIIA, which alters function, associates with SLE phenotype, J. Invest. Med. 45:200A [1997]; R. Mehrian et al., Synergistic effect between IL-10 and Bcl-2 genotypes in determining susceptibility to systemic lupus erythematosus, Arthritis Rheum. 41:596–602 [1998]). Mutations in nucleic acids encoding T cell receptor zeta chain have been linked to SLE in some patients. (K. Tsuzaka et al., Mutations in T cell receptor zeta chain mRNA of peripheral T cells from systemic lupus erythematosus patients, J. Autoimmun. 11(5):381–85 [1998]). Some candidate genes may confer risk only to subsets of SLE patients. For example, FcγRIIA alleles (the gene encoding a 40-kD FcγR expressed on human mononuclear phagocytes and neutrophils) confer an increased risk for lupus GN in African Americans, but not in Caucasians, or persons of Afro-Caribbean or Chinese origin. (J. E. Salmon et al., FcγRIIA alleles are heritable risk factors for lupus nephritis in African-Americans, J. Clin. Invest. 97:1348–54 [1996]; M. Botto et al., FcγRIIA polymorphism in systemic lupus erythematosus [SLE], Clin. Exp. Immunol. 104:264–68 [1996]).
Elements that complicate the study of disease-causing genes in genetically complex diseases, such as human SLE, include ethnic diversity, clinical heterogeneity (and presumably genetic heterogeneity), reduced penetrance (genetic expressivity), and the effect of environment (E. S. Lander and N. J. Schork, Genetic dissection of complex traits, Science 265:2037–48 [1994]). In contrast, murine models of spontaneous lupus in inbred strains are less complex, and consequently murine models of disease susceptibility provide a more accessible route for investigating genetically-linked disease in humans. Recent success in mapping a susceptibility locus for multiple sclerosis in the 5p14–p12 region, which is syntenic to the murine locus Ea2, further supports the utility of this mouse-to-human approach. (S. Kuokkanen et al., A putative vulnerability locus to multiple sclerosis maps to 5p14–p12 in a region syntenic to the murine locus Eae2, Nature Genet. 13:477–80 [1996]).
Genetic studies of murine SLE have identified susceptibility loci in several inbred strains which spontaneously develop SLE GN. (Reviewed in A. N. Theofilopoulus, The basis of autoimmunity: Part II. Genetic predisposition, Immunology Today 15:150–58 [1995]). These studies have included genome-wide searches for evidence of linkage using backcrosses or F2 intercrosses of lupus mice such as MRL/LPR, NZB/NZW and NZM/Aeg2410 (M. L. Watson et al., Genetic analysis of MRL-lpr mice: Relationship of the Fas apoptosis gene to disease manifestations and renal disease-modifying loci, J. Exp. Med. 176:1645–56 [1992]; D. H. Kono et al., Lupus susceptibility loci in New Zealand mice, Proc. Natl. Acad. Sci. USA 91;10168–72 [1994];Drake et al., Analysis of the New Zealand Black contribution to lupus-like renal disease: multiple genes that operate in a threshold manner, J. Immunol. 154:241–47 [1995]; Drake et al., Genetic analysis of the NZB contribution to lupus-like autoimmune disease in [NZB×NZW]F1 mice, Proc. Natl. Acad. Sci. USA 91:4062–66 [1994]; S. Hirose et al., Mapping of a gene for hypergammaglobulinemia to the distal region chromosome 4 in NZB mice and its contribution to systemic lupus erythematosus in [NZB×NZW]F1 mice, Internat. Immmunol. 12:1857–64 [1994]; L. Morel et al., Polygenic control of susceptibility to murine SLE, Immunity 1:219–229 [1994]). Four genomic intervals strongly linked to GN in mice derived from different parental strains have been identified in multiple studies at loci on chromosomes 1, 4, 7, and 17. The distal end of mouse chromosome 1 was shown to predispose to specific manifestations of SLE, including GN, IgG anti-chromatin antibodies, and splenomegaly. (D. H. Kono et al. [1994]; Drake et al. [1995]; L. Morel et al. [1994]).
There is evidence for a corresponding genetic linkage in human SLE. Using the identified murine susceptibility loci (the overlapping SLE/Nba2/Lbw7) as a guide, Tsao et al. examined seven markers located on a syntenic human chromosomal 1q31–q42 region, corresponding to the telomeric end of mouse chromosome 1, the latter being the region where contributions to specific manifestations of murine lupus, including glomerulonephritis and IgG anti-chromatin, have been mapped. (B. Tsao et al., Evidence for linkage of a candidate chromosome 1 region to human systemic lupus erythematosus, J. Clin. Invest. 99:725–731 [1997]). The seven markers were examined in 52 affected human sibpairs from Caucasian, Asian and African-American families. Five markers located in a 15 cM region of human chromosome 1q41–q42 showed evidence for linkage excessive by the allele sharing method (B. Tsao et al. [1997]; B. Tsao et al., The genetic basis of systemic lupus erythematosus, Proc. Assoc. Am. Physicians 110(2): 113–17 [Review March-April 1998]). Subsequently, an independent sample of 105 SLE-affected sibpairs also supported linkage of the 1q41–q42 region with SLE. (P. M. Gaffney et al., A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families, Proc. Natl. Acad. Sci. USA [in press]).
The distance (>60 cM) between the 1q41–q42 region and the FcγRIIa gene (1q23) make it unlikely that this gene could account for the observed linkage between the 1q41–q42 region and SLE. A polymorphism for low expression of CR1 (complement receptor one, previously C3b/C4b receptor) has been suggested to be a risk factor for SLE (P. Cornillet et al., Increased frequency of the long (S) allotype of CR1 (the C3b/C4b receptor, CD35) in patients with systemic lupus erythematosus, Clin. Exp. Immunol. 89:22–25 [1992]), although a later study suggests that low CR1 expression is acquired. (A. Kumar et al., HindIII genomic polymorphism of the C3b receptor (CR1 in patients with SLE: low erythrocyte CR1 expression in acquired phenomenon, Immunol. Cell Biol. 73:457–62 [1995]). However, the CR1 gene maps to chromosome 1q32, and linkage disequilibrium at the 1q41–42 region, but not at the 1q31–32 region, make CR1 an unlikely candidate as a susceptibility gene for SLE. (B. Tsao et al. [1997]).
Within the human chromosomal 1q41–q42 region, there are three candidate genes for linkage with an SLE phenotype. One of these, HLX1, is expressed in hematopoietic progenitors and activated lymphocytes and encodes a homeo box protein which may regulate the development of CD4+ T-cells. (Y. Deguchi et al., A human homeobox gene, HB24, inhibits development of CD4+ T cells and impairs thymic involution in transgenic mice, J. Biol. Chem. 268:3646–53 [1993]). Another, TGFB2 (transforming growth factor beta-2) can suppress Il-2 dependent T-cell growth (R. Demartin et al., Complementary DNA for human glioblastoma-derived T cell suppressor factor, a novel member of the transforming growth factor-beta gene family, EMBO J. 6:3673–77 [1987]), and administration of a TGFβCDNA expression vector to MRL/lpr mice decreases serum IgG anti-chromatin and delays the onset of SLE. (E. Raz et al., Modulation of disease activity in murine systemic lupus erythematosus by cytokine gene delivery, Lupus 4:286). However, TGFB2 is not expressed in hematopoietic cells, and such expression is believed to be an essential link in the etiology of SLE.
The third candidate gene, PARP, sometimes referred to as ADPRT (ADP-ribosyltransferase gene), is induced by DNA damage and plays a role in cellular repair (P. Loetscher et al., Poly(ADP-ribose) may signal changing metabolic conditions to the chromatin of mammalian cells, Proc. Natl. Acad. Sci. USA 84:1286–89 [1987]). Haug et al. reported that SLE patients and their family members have decreased poly(ADP-ribose) metabolism. (B. L. Haug et al., Altered poly(ADP-ribose) metabolism in family members of patients with systemic lupus erythematosus, J. Rheum. 21:851–56 [1994]), which is evidence that PARP is linked to SLE.
The PARP gene encodes poly(ADP-ribosyl)transferase, also known as poly(ADP-ribosyl)polymerase, which is a zinc-finger DNA-binding protein that repairs DNA damage and is specifically cleaved during apoptosis. (B. Tsao et al., ADPRT alleles from the chromosome 1q41–q42 linked region are associated with SLE, Arthritis & Rheumatism 41(9)(Suppl.):283 [Abstract, Sept. 1998]). In their abstract, Tsao et al. reported that none of the tested alleles of HLX1 showed evidence for association with SLE phenotypes in a family-based linkage test applied to 124 families. However, a polymorphism within the PARP region was associated with SLE phenotype. This polymorphism was reported to be a dinucleotide repeat in the PARP promoter region.
There has been a need for a rapid and accurate genetic test for SLE. Previously available diagnostic tests for SLE are limited in their diagnostic effectiveness. Rheumatoid factors (RF), antinuclear antibodies (ANA), and erythrocyte sedimentation rate (ESR) are among the most frequently used tests for the diagnosis and investigation of connective tissue disease, including SLE. However, positive predictive values for these diagnostic tests are reported to be low as to a diagnosis of SLE, albeit they may be more predictive for a subject having other clinical SLE symptoms from among the 11 revised SLE criteria of the American College of Rheumatology (ACR), such as a typical malar or discoid rash, photosensitivity, oral ulcers, arthritis, serositis, or disorders of blood, kidney or nervous system. (E. M Tan et al., The 1982 revised criteria for the classification of systemic lupus erythematosus [SLE], Arthritis Rheum. 25:1271–77 [1982]; M. E. Suarez-Almazor et al., Utilization and predictive value of laboratory tests in patients referred to rheumatologists by primary care physicians, J. Rheumatol. 25(10);1980–85 [1998]; C. A. Slater et al., Antinuclear antibody testing. A study of clinical utility, Arch. Intern. Med. 156(13): 1421–25[1996]). The diagnostic utility of ELISA assay for the presence of anti-extractable nuclear antigen (anti-ENA) antibodies is limited to those patients that also test positive for both ANA and anti-dsDNA antibodies. (J. Sanchez-Guerrero et al, Utility of antiSm, anti-RNP, anti-Ro/SS-A, and anti-La/SS-B [extractable nuclear antigens] detected by enzyme-linked immunosorbent assay for the diagnosis of systemic lupus erythematosus, Arthritis Rheum. 39(6):1055–61 [1996]).
Unfortunately, a patient's presentation of clinical symptoms in SLE may be vague or idiopathic, easily mistaken for another disorder. For example, uncommon clinical manifestations associated with underlying SLE may include, acute pancreatitis, pure red cell aplasia, cystitis of the urinary tract, antiphospholipid antibody syndrome, or neurological complications of normal pressure hydrocephalus. (K. P. Leong and M. L. Boey, Systemic lupus erythematosus [SLE] presenting as acute pancreatitis—a case report, Singapore Med. J. 37(3):323–24 [1996]; M. H. Tsai et al., Systemic lupus erythematosus with pure red cell aplasia: a case report, Chung Hua I Hsueh Tsa Chih [Taipei] 52(4):265–68 [1993]; Y. Nakauchi et al., Systemic lupus erythematosus relapse with lupus cystitis, Clin. Exp. Rheumatol. 13(5):645–48 [1995]; P. R. Mortifee et al., Communicating hydrocephalus in systemic lupus erythematosus with antiphospholipid antibody syndrome, J. Rheumatol. 19(8):1299–1302 [1992]; H. Y. You et al., Normal pressure hydrocephalus in a patient with systemic lupus erythematosus: a case report, Chung Hua I Hsueh Tsa Chih [Taipei] 61(9):551–55 [1998]; M. D. Uhl et al., Normal pressure hydrocephalus in a patient with systemic lupus erythematosus, J. Rheumatol. 17(2): 1689–91 [1990]).
Consequently, a genetic testing method for SLE has been needed that can be used in conjunction with other diagnostic tests for SLE. This, and other advantages described herein, the present invention provides.